Structural characterization of phosphatidylinositol 4-phosphate IIa

Adriana Baumlová, Evžen Boura, Dominika Chalupská

Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo nam. 2, 166 10 Praha 6

Phosphoinositides are essential regulators of fundamental cellular processes. Phosphatidylinositol 4-kinases (PI4K) initiate the canonical phosphoinositide biosynthetic pathway by phosphorylating D-4 hydroxyl of the inositol head group. Two types of human PI4K, designated as type II and III, are distinguished. The biological role of PI4K type IIα is diverse. PI4K IIα generates PtdIns4P pools and thus modulates biological membrane composition, membrane trafficking, endocytosis or signal transduction. Moreover, PI4K IIα is involved in Wnt signaling that regulates embryonic and bone development, tumorigenesis, neurogenesis and lipid and glucose metabolism. PI4K IIα also represents the important regulatory molecule of lysosomal β-glucocerebrosidase delivery. Depletion of PI4K IIα results in Gaucher disease, lysosomal storage disorder caused by a defect in the degradation of glucosylceramide.

Significant biological relevance of PI4K IIα, its insufficient structural characterization and the fact that PI4K IIα doesn't share sequence homology with any other crystallized kinase lead us to its structural characterization. In order to increase the solubility of PI4K IIα, the sequence of T4 lysozyme was fused within this protein. It´s quite common method used for membrane protein crystallization. PI4K IIα with T4 lysozyme was expressed in Escherichia  coli and purified using affinity chromatography and size exclusion chromatography. Obtained protein with high purity was used for crystallization trials. PI4 IIα crystallized in orthorhomboic space group P 2₁22₁ (a = 79.55 Å, b = 78.71 Å, c = 104.75 Å, α = β = γ = 90°). Native crystals diffracted to resolution 2.8 Å. The phase problem was solved by MR SAD using SeMet crystals and the T4 lysozyme as a search model.